Microfluidics is revolutionizing the way activities are performed in a substantial proportion of chemical and physical operations. One area of microfluidics is the manipulation of small volumes of liquids or liquid compositions on a solid substrate, where a network of channels and reservoirs are present. By employing electric fields with electrically conducting liquids, volumes and/or ions can be moved from one site to another, different solutions formed by mixing liquids and/or ions, reactions performed, separations performed, and analyses carried out. In fact, in common parlance, the system has been referred to as “a laboratory on a chip.” Various prior art devices of this type include U.S. Pat. Nos. 6,010,608, 6,010,607, 6,001,229, 5,858,195, and 5,858,187 which are a family of applications concerned with injection of sample solutions. See also, U.S. Pat. No. 5,599,432, European Pat. No. 0620432, international patent application no. WO 01/59440, and Verheggen et al., J. of Chromatography 452 (1988) 615-622.
In many of the operations, there is an interest in electrophoretically separating multiple sample components contained in dilute samples. For example, U.S. Patent Application Pub. No. 2002/0142329 describes a technique for performing assays for multiple target molecules using electrophoretic tag (e-tag) probes. The e-tag probes interact with a target, such as a single-stranded nucleic acid, a protein, a ligand-binding agent, such as an antibody or receptor, or an enzyme, e.g., as an enzyme substrate. The e-tag probes include a “portion” that binds to the target. After the target-binding portion of an e-tag probe binds to a target, a linking group of the electrophoretic probe is cleaved to release an “electrophoretic tag” that has a unique mass or charge-to-mass ratio, rendering such e-tags separable by, for example, electrophoretic separation on microfluidic devices.
Various processes are available for carrying out electrophoretic separations including but not limited to zone or capillary electrophoresis (CE) and isotachophoresis (ITP). Briefly, CE separations are performed in a capillary or channel filled with an electrophoretic medium under the influence of an electric field. Particles of different sizes and charges migrate along the channel at different speeds such that different components of a sample tend to separate spatially.
ITP processes are different than CE separations in that an ITP process stacks components of the sample between two electrolytes—a leading electrolyte and a trailing electrolyte. The components of the sample form bands between trailing and leading electrolytes. This phenomenon has been described in, e.g., Everaerts et al, Isotachophoresis. Theory, Instrumentation and Applications (Elsevier, Amsterdam, 1976); Burgi and Chien, chapter 16, in Landers, editor, Handbook of Capillary Electrophoresis, Second Edition (CRC Press, Boca Raton, 1997). See also international patent application no. WO 01/59440.
FIGS. 1A-1B illustrate an ITP separation. Referring to FIGS. 1A and 1B, a sample 58 containing components with different electrophoretic mobilities is placed between an electrolyte 60 with a leading edge ion and an electrolyte 62 containing a terminating or trailing-edge ion. These components may be placed in a capillary channel or tube, a section of which is shown at 56. The leading edge ion is a small ion, such as the chloride ion, having an electrophoretic mobility greater than that of any of the sample components. The counter-ion of the leading-edge ion is preferably chosen for its ability to buffer the solution.
Similarly, the trailing edge ion is one having an electrophoretic mobility lower than the slowest-migrating sample components. With the application of a voltage potential across the sample, sample components will band, by migration through the sample, until the fastest moving sample components are concentrated adjacent the leading-edge electrolyte and the slowest moving components, against the trailing edge electrolyte.
In the figures, the sample components to be separated are negatively charged, as are the leading- and trailing-ions, and the polarity of voltage is applied with the polarity shown, to attract the negatively charged components toward the right in the figures. Because the electric field across each section of the system is inversely proportional to the conductivity in that section, the section associated with the leading-edge ion is characterized by a relatively low electric field, and the section associated with the trailing-edge ion, with a relatively high electric field.
It is this different electric field or voltage gradient that maintains the sample components in a narrow band of sample components, each separated on the basis of their electrophoretic mobilities, once the sample components have stacked into a narrow band. Sample ions that diffuse back into the trailing electrolyte “speed up” under the higher electric field. Similarly, sample ions that diffuse forward into the leading electrolyte slow down under the lower electric field. At the same time, each sample component migrates to a position closely adjacent the sample components nearest in electrophoretic mobility, causing the components to stack into a tight sample band of separated components between the leading- and trailing-ion electrolytes.
Although each electrophoretic process affects the migration of the individual components of a sample, ITP can increase sensitivity by concentrating analytes. CE, on the other hand, though typically providing adequate sensitivity provides increased resolution of analytes having different mobilities.
U.S. Pat. App. 2002/0079223 describes an ITP-CE zone separation process. A combined ITP-CE separation process has attributes of each of the separation processes resulting in improved spatial separation between the analytes and improved efficiency. Briefly, as will be described further below, a combined ITP-CE separation first stacks the sample between the trailing edge electrolyte and the leading edge electrolyte to form component bands. Once the stack is formed, the stack is spatially separated along a channel to improve the spatial separation between the analytes.
In carrying out any of the above described electrophoretic separation processes on a microfluidic device, it is not surprising that voltage control is critical. Fluid and sample manipulation are controlled by application of voltages. However, accurately timing the application of voltages is fraught with difficulty since the timing depends on, amongst other things, the sample to be assayed. Indeed, the mobility of the sample ions is affected by a number of factors including, for example, viscosity of the liquids.
Various techniques for timing and application of voltages to drive the electrophoretic processes include a.) observing separation results and adjusting the timing and voltages based on the observed results and b.) visualizing dye concentrations under a microscope and adjusting the timing and voltages based on the microscope observations. Each of these techniques has marked drawbacks. Observation and analyzing separation results requires testing and optimization for each sample prior to commencing the actual testing. This is impractical.
Visualizing dye concentrations is also undesirable. Visualization of dye concentrations is undesirable because it is based on operator (human) observation. Activation of voltages or voltage switching is performed after a subjective observation of a microfluidic event such as, e.g., observing a stacked sample crossing a point along the channel. Human observation may be inaccurate and vary with time. Over long periods of time, for example, the observer's senses may deteriorate due to fatigue.
It is therefore desirable to provide an improved technique for controlling the application of voltages to carry out electrophoretic operations and other types of procedures on microfluidic devices. It is desirable to provide a technique that controls the voltages to drive electrophoretic separations on microfluidic devices and that does not suffer from the above mentioned drawbacks.